Treatment of acute coronary syndrome with an exendin

ABSTRACT

The invention relates to methods for treating a patient suffering from acute coronary syndrome, but who is not suffering from a Q-wave myocardial infarction, comprising administration of a therapeutically effective amount of a GLP-1 molecule. The GLP-1 can be self-administered, and can be administered in one or more doses, as needed, on an intermittent or continuous basis, to optimize metabolism in cardiac tissue and to prevent cardiac damage associated with ischemia.

BACKGROUND OF THE INVENTION

Heart disease is a major health problem throughout the world. Myocardialinfarctions are a significant source of mortality among thoseindividuals with heart disease.

Acute coronary syndrome (“ACS”) denotes patients who have or are at highrisk of developing an acute myocardial infarction (MI). This complexincludes unstable angina (UA), non-Q-wave cardiac necrosis (NQCN) andQ-wave MI (QMI). Thompson et al., M.J.A. 171; 153 (1999). Typically, ACSis diagnosed when a patient has acute (i.e., sudden onset) chest pain ofa cardiac origin that is either new or clearly different frompre-existing, chronic, stable angina; that is, ACS chest pain is moresevere, more frequent, occurs at rest, or is longer than 15 minutes induration. After ACS has been diagnosed, the patient is stratified intoUA, NQCN, and QMI, using criteria that are described elsewhere in thisapplication. UA, NQCN, and QMI are believed to represent differentstages of plaque rupture and thrombosis. Zaacks et al., J. Am. CollegeCardiol. 33; 107 (1999). With UA, there typically is no myocardialnecrosis. Id. UA, NQCN, and QMI all are characterized by varying degreesof ischemia. Id. Additionally, Q-wave MI generally is understood toresult from total occlusion of a coronary artery, whereas UA is causedby a subtotal occlusion. Thompson et al. M.J.A. 171; 153 (1999).

During normal, aerobic metabolism, cardiac tissue uses free fatty acids(FFA) to generate energy. During ischemia induced by UA, NQCN, or Q-waveMI, the heart switches to anaerobic metabolism, using glucose as itsprimary energy source.

Many other detrimental metabolic changes occur during ischemia incardiac tissue, including accumulation of excess unoxidized FFAproducts, inhibition of Ca²⁺ and Na⁺/K⁺ pumps, and increased levels ofcAMP. Additionally, there is decreased secretion of insulin bypancreatic β-cells and excess secretion of glucagon by pancreaticα-cells.

Excess glucagon can lead to myocardial tissue damage; glucagon is alsoan insulin antagonist and mediates lipolysis in adipose tissue, withrelease of FFAs. Excess FFAs can lead to free radical formation andconsequent tissue damage. Glucagon is one of the so-calledcounter-regulatory hormones, a group that includes cortisol, growthhormone, and catecholamines, which are released during “stress”conditions, such as ACS, UA, NQCN, fasting, starvation, infection,disease, internal injury, and trauma. The role of such hormones is tocounter-regulate the effects of insulin, thereby raising blood glucoseand fatty acid levels and producing a generally insulin-antagonisticstate. Glucose is a mediator of stress responses and a component ofsystemic inflammatory reactions.

A variety of therapeutic agents is known for treating Q-wave MI. Theseinclude thrombolytic therapy and angiotensin-converting enzyme (ACE)inhibitors. Thompson et al., M.J.A. 171; 153 (1999). PCT Application WO98/08531 relates to treatment with GLP-1 of a patient suffering fromQ-wave MI who is also incapable of auto-regulation of blood glucose.

Agents known for treatment of a subtotal coronary occlusion, whichresults in UA, include heparin, low-molecular-weight heparin, andnitroglycerine. Thompson et al., M.J.A. 171; 153 (1999). β-blockers canbe used to combat myocardial ischemia and left ventricular dysfunctionthat result from acute MI and UA. Id. Prior to the formation of a fibrinthrombus, which leads to partial or total coronary artery occlusion, itis known that that there is plaque erosion or fissure, followed byplatelet aggregation. This aggregation can be treated with aspirin,glycoprotein IIb/IIIa antagonists or clopidogrel. Thompson et al.,M.J.A. 171; 153 (1999).

Most therapies for the treatment of UA work by (1) stabilizing orreducing the occlusion, such as the anti-thrombin agents heparin andlow-molecular-weight heparin, and the anti-platelet agents aspirin,glycoprotein IIb/IIIa antagonists, or clopidogrel, (2) reducing preload,such as nitroglycerine, (3) reducing afterload, such as ACE inhibitors,or (4) reducing myocardial oxygen demand, such as β-blockers. Thesetherapies do not treat directly the disturbed energy metabolism thatresults from ischemia and that induces tissue damage. Also, dosages ofdrugs such as heparin must be controlled carefully to avoid toxiceffects of overdose.

As a result, there is a need for therapeutic treatments that can be usedpreferably beginning at the earliest stages of ACS, and during UA orNQCN, and that will prevent and/or reduce the damage resulting from ACS,including any subsequent Q-wave MI.

SUMMARY OF THE INVENTION

Objects of the present invention include the following:

(1) A method of treating a patient suffering from acute coronarysyndrome, comprising administering to the patient a therapeuticallyeffective amount of a GLP-1 molecule, wherein the patient is notsuffering from a Q-wave MI. The above method, wherein the patient issuffering from unstable angina. The above method, wherein the patient issuffering from non-Q-wave cardiac necrosis. The above method, whereinthe patient has a blood troponin I level of no more than 0.4 ng/ml. Theabove method, wherein the patient has a blood troponin T level of nomore than 0.1 ng/ml. The above method, wherein the patient does not haveelevated blood creatine kinase. The above method, wherein the patientdoes not have ST-segment elevation. The above method, wherein thepatient does not exhibit a pathological Q-wave. The above method,wherein the patient exhibits one or more of the following symptoms:chest rain greater than 15 minutes in duration, chest pain at rest, orchest pain following minimal exertion that is poorly responsive tosublingual nitrates. The above method, wherein the patient has stableangina. The above method, wherein the patient administers the GLP-1 tohimself. The above method, wherein the GLP-1 is administered in the formof a GLP-1-stick. The above method, wherein the GLP-1 is administered ina single dose. The above method, wherein the GLP-1 is administered inmore than one dose. The above method, wherein the GLP-1 is administeredcontinuously. The above method, wherein glucose, or a potassium salt, ora combination thereof, is co-administered with the GLP-1.

(2) A method for treatment of a patient, comprising administering to theindividual a therapeutically effective amount of a GLP-1 molecule,wherein the administration is after the onset of one or more of thefollowing symptoms: chest pain lasting longer than 15 minutes, chestpain at rest, chest pain following minimal exertion, nausea, shortnessof breath, palpitations, or dizziness. The above method, wherein thepatient has not suffered a Q-wave MI prior to the onset of the symptomor symptoms. The above method, wherein the patient is suffering fromunstable angina. The above method, wherein the patient is suffering fromnon-Q-wave cardiac necrosis. The above method, wherein the patient has ablood troponin I level of no more than 0.4 ng/ml. The above method,wherein the patient has a blood troponin T level of no more than 0.1ng/ml. The above method, wherein the patient does not have elevatedblood creatine kinase myocardial isoenzyme. The above method, whereinthe patient does not have ST-segment elevation. The above method,wherein the patient does not exhibit a pathological Q-wave. The abovemethod, wherein the administration occurs between the time of onset ofthe one or more symptoms, and the time the patient suffers a Q-wave MI.The above method, further comprising the step of continuing theadministration of a GLP-1 molecule during the time that the patientsuffers a Q-wave MI. The above method, further comprising the step ofcontinuing the administration of a GLP-1 molecule after the time thepatient suffers a Q-wave MI. The above method, wherein the patient hasischemic heart disease, or is at risk for developing ischemic heartdisease. The above method, wherein the patient has one or more of thefollowing cardiac abnormalities: congestive heart failure, worseningheart murmur due to mitral regurgitation, or evidence of cardiacconduction disturbances. The above method, wherein the patient has anormal ECG. The above method, wherein the patient has stable angina. Theabove method, wherein the patient administers the GLP-1 to himself. Theabove method, wherein the GLP-1 is administered in the form of aGLP-1-stick. The above method, wherein the GLP-1 is administered in asingle dose. The above method, wherein the GLP-1 is administered in morethan one dose. The above method, wherein the GLP-1 is administeredcontinuously. The above method, wherein glucose, or a potassium salt, ora combination thereof, is co-administered with the GLP-1.

(3) A method for treating a patient suffering from stable angina,comprising administration of a GLP-1 molecule. The above method, whereinthe administration is continuous.

(4) A method for performing angioplasty on a patient in need thereof,comprising administering a GLP-1 molecule to the patient during theangioplasty procedure. The above method, further comprisingadministering a GLP-1 molecule to the patient prior to the angioplastyprocedure. The above method, further comprising administering a GLP-1molecule to the patient following the angioplasty procedure.

(5) A method for treatment of a patient with ischemic heart disease, oris at risk for developing ischemic heart disease, and who exhibits oneor more of the following symptoms: nausea, shortness of breath,palpitations, or dizziness, and further wherein the patient does notexhibit chest pain, comprising administering to the patient atherapeutically effective amount of a GLP-1 molecule, wherein thepatient is not suffering a Q-wave MI. The above method, wherein thepatient has a normal ECG.

(6) A method for increasing the time during which thrombolytic therapywill be effective following the first symptom of cardiac distress,comprising administering a therapeutically effective amount of a GLP-1molecule after the onset of one or more of the following symptoms: chestpain lasting longer than 15 minutes, chest pain at rest, chest painfollowing minimal exertion, nausea, shortness of breath, palpitations,or dizziness.

(7) A kit comprising one or more doses of a GLP-1 molecule, the kitcomprising a device selected from the group consisting of aninsulin-type syringe, a “pen” injector that delivers a metered dose, aneedle-less injector, a liquid-formulation, a dry-powder inhaler, abuccal tablet, and a sublingual tablet.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Thus, the invention encompasses novel methods for the treatment of acutecoronary syndrome, particularly unstable angina and non-Q-wave cardiacnecrosis, with a GLP-1 molecule. The methods of the present inventioncan be used beginning at the earliest stages of ACS, prior todevelopment of a Q-wave MI, to prevent damage associated with ischemiathat occurs during Q-wave MI. The inventive therapeutic methods that usea GLP-1 molecule reverse or ameliorate the ischemia-induced damage thatoccurs during UA and NQCN. The methods of the invention further comprisecontinuing the treatment with a GLP-1 molecule during or after the timethat a patient suffers from a QMI.

Definitions

As used in this application, a “Q-wave MI” denotes a condition that isdiagnosed in a patient who exhibits a pathological Q-wave, as indicatedby electrocardiogram (ECG), and who has one or more of the followingsymptoms and signs: (1) ST elevation, as measured b. ECG; (2) elevatedblood levels of troponin I and troponin T, associated with a Q-wave MI;(3) elevated blood creatine kinase myocardial isoenzyme (CK-MB) level,associated with a Q-wave MI; and (4) elevated blood lactatedehydrogenase level, associated with a Q-wave MI. Typically, thepathological Q-wave will be exhibited within about 6-18 hours of a totalcoronary occlusion. The skilled artisan will understand that a diagnosisof Q-wave MI generally indicates the presence of a totally occludedcoronary artery. Furthermore, the skilled artisan will understand thatthe diagnosis of QMI is one of medical judgment.

“Elevated” troponin I levels of greater than 0.4 ng/ml typically arehighly predictive of some degree of Q-wave MI. Antman et al., New Engl.J. Med., 335; 1342 (1996). “Elevated” troponin T levels of greater than0.1 ng/ml typically are highly predictive of some degree of Q-wave MI.Ohman et al., New Engl. J. Med., 335; 1333 (1996). Increased troponinlevels can be observed within about 2 hours of a QMI. See, e.g.,Klootwijk et al., A.C.S. 353, 10 (1999); U.S. Pat. No. 5,690,103.

“Elevated” CK-MB levels of greater than 10 U/liter and greater than 5%of total CK enzyme activity typically are highly predictive of somedegree of Q-wave MI. Thompson et al., M.J.A. 171; 153 (1999). IncreasedCK-MB levels can be observed within about 3 to 4 hours after QMI. See,e.g., U.S. Pat. No. 5,690,103.

One example of “elevated” LDH levels that are associated with a QMI is asignificant rise in LDH and at least one plasma sample showing LDH1levels greater than LDH2 levels. Furlong et al., Clin. Chem. 96; 134(1991). Another example of such elevated levels is two serum values ofLDH that are at least two standard deviations above the normal range.Malmberg et al., J. Am. Col. Cardiol. 26; 57 (1995).

Q-wave MI is typically accompanied by chest pain of at least 15 minutesin duration. However, diagnosis of chest pain alone does not indicatethat the patient is suffering from Q-wave MI.

The diagnostic criteria for UA and NQCN are quite different from thosefor Q-wave MI, although all can be characterized by chest pain. As usedherein, a patient suffering from “unstable angina” denotes a patient whohas one or more of the following symptoms and signs: (1) ST segmentdepression, as measured by ECG; (2) slightly elevated troponin T levels,of no more than 0.1 ng/ml; or (3) slightly elevated troponin I levels,of no more than 0.4 ng/ml. In contrast to Q-wave MI, CK-MB and LDHlevels are typically not elevated during UA. Also in contrast to Q-waveMI, a patient with UA typically has no ST segment elevation nor anypathological Q-wave. Finally, UA can be diagnosed solely on the basis ofchest pain, typically chest pain lasting longer than 15 minutes, chestpain at rest, or chest pain following minimal exertion and that ispoorly responsive to sublingual nitrates. Alternatively, even in theabsence of chest pain, a patient can be diagnosed with UA if previouslydiagnosed with ischemic heart disease (IHD) or is considered to be atstrong risk for developing IHD, and who presents with nausea, shortnessof breath, palpitations, or dizziness. Furthermore, the skilled artisanwill understand that the diagnosis of UA is one of medical judgment.

As used herein, “ischemic heart disease” denotes disease of cardiactissue that results from a decreased oxygen supply to the cardiac tissuethat is due to reduced coronary artery blood flow. Typically, thisreduced blood flow results from the partial or complete obstruction ofblood vessels that service the heart. A diagnosis of IHD can be based onthe presence of chronic, stable angina, elicited by exercise (also knownas “exertional angina”) that is relieved by sublingual nitrates. Adiagnosis of IHD also can be based on an ECG reading that is consistentwith IHD, such as one exhibiting ST segment deviations and/or T waveinversions.

NQCN can present similarly to UA. As used herein, a patient sufferingfrom “non-Q wave cardiac necrosis” denotes a patient who does not have apathological Q-wave, but who has one or more of the following symptomsand signs: (1) ST segment elevation or depression, as measured by ECG,(2) elevation of troponin I, greater than 0.4 ng/ml; (3) elevation oftroponin T, greater than 0.1 ng/ml; or (4) elevation of CK-MB greaterthan 10 U/liter within 2448 hours of onset of symptoms. Typically, aNQCN patient will present with chest pain of cardiac origin lastinglonger than 15 minutes, with or without ST segment elevation ordepression. Furthermore, the skilled artisan will understand that thediagnosis of NQCN is one of medical judgment.

“Angina” or “angina pectoris” generally refers to chest pain resultingfrom an insufficient blood supply to the heart. Angina pectoris is arecurring symptom and usually occurs in the form of chest discomfort(tightness, fullness, squeezing, heaviness, burning or pain) in thecenter of the chest and/or over the left breast. The discomfort may moveto the left shoulder and arm, although it may move to bothshoulders/arms, throat, jaw, or even the lower portion of the chest orupper abdomen. It may be accompanied by shortness of breath, sweating,weakness, dizziness, nausea, or numbness in the shoulders, arms, orhands. Symptoms of angina pectoris are typically triggered by physicalexertion. The symptoms are generally brief, last only 2-3 minutes andsubside promptly with cessation of exercise or following the use of anitroglycerin tablet, which typically is administered via a sublingualroute. This pattern of pain is known as “stable angina.” “Chronic stableangina” generally is used to describe a patient who routinely exhibitsthe symptoms of “stable angina” over a prolonged period of weeks,months, or years.

As used herein “symptom of cardiac distress” is used to denote one ormore of the following: chest pain lasting longer than 15 minutes, chestpain at rest, chest pain following minimal exertion, nausea, shortnessof breath, palpitations, or dizziness. The skilled artisan willrecognize that palpitations generally are recognized by a “racing”heartbeat, beating more quickly than normal, during rest conditions.

As used herein, a “GLP-1 molecule” includes the following. Mammalian GLPpeptides and glucagon are encoded by the same gene. In the ileum theprecursor is processed into two major classes of GLP peptide hormones,namely GLP-1 and GLP-2. GLP-1(1-37) has the sequence: His Asp Glu PheGlu Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu GlyGln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly (SEQ IDNO:1). GLP-1 (1-37) is amidated by post-translational processing toyield GLP-1(1-36)NH₂, which has the sequence: His Asp Glu Phe Glu ArgHis Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln AlaAla Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg (NH₂) (SEQ ID NO:2), oris enzymatically processed to yield GLP-1(7-37), which has the sequence:His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln AlaAla Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly (SEQ ID NO:3).GLP-1(7-37) can also be amidated to yield GLP-1(7-36)amide, which is thenatural form of the GLP-1 molecule, and which has the sequence: His AlaGlu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala LysGlu Phe Ile Ala Trp Leu Val Lys Gly Arg (NH₂) (SEQ ID NO:4). Likewise,GLP-1(1-36)amide can be processed to GLP-1(7-36)amide.

Intestinal L cells secrete GLP-1(7-37) (SEQ ID NO:3) and GLP-1(7-36)NH2(SEQ ID NO: 4) in a ratio of 1 to 5. These truncated forms of GLP-1 haveshort half-lives in vivo (less than 10 minutes), and are inactivated byan aminodipeptidase IV to yield: Glu Gly Thr Phe Thr Ser Asp Val Ser SerTyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly ArgGly (SEQ ID NO:5), and Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr LeuGlu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg (NH₂)(SEQ ID NO:6), respectively. It has been speculated that the peptidesGlu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala LysGlu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly (SEQ ID NO:5) and Glu GlyThr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu PheIle Ala Trp Leu Val Lys Gly Arg (NH₂) (SEQ ID NO:6) affect hepaticglucose production, but do not stimulate production or release ofinsulin from the pancreas.

As used in this specification, the term “GLP-1 molecule” includesGLP-1(1-37), GLP-1(1-36)NH₂, GLP-1(7-37), GLP-1(7-36)NH2 (“GLP-1(7-36)amide”) (collectively referred to as “GLP-1 peptides”). The presentinvention includes the use of recombinant human GLP-1 peptides and GLP-1peptides derived from other species, whether recombinant or synthetic.

“GLP-1 molecule” further denotes biologically active variants, analogs,and derivatives of GLP-1 peptides. “Biologically active,” in thiscontext, means having GLP-1(7-36) biological activity, but it isunderstood that the variant, analog, or derivative can be either less ormore potent than native GLP-1(7-36)amide, a native, biologically activeform of GLP-1. See Goke & Byrne, Diabetic Medicine. 13; 854 (1996).GLP-1 molecules of the present invention include polynucleotides thatexpress agonists of GLP-1 (i.e., activators of the GLP-1 receptormolecule and its secondary messenger activity found on, inter alia,insulin-producing β-cells). GLP-1 mimetics that also are agonists ofβ-cells include, for example, chemical compounds specifically designedto activate the GLP-1 receptor.

Included as GLP-1 molecules are any molecules, whether they be peptides,peptide mimetics, or other molecules that bind to or activate a GLP-1receptor, such as the GLP-1(7-36)amide receptor, and its secondmessenger cascade. GLP-1 molecules include species having insulinotropicactivity and that are agonists of (i.e., activate), the GLP-1 receptormolecule and its second messenger activity on, inter alia, insulinproducing β-cells.

“GLP-1 molecules” also include peptides that are encoded bypolynucleotides that express biologically active GLP-1 variants, asdefined herein. Also included in the present invention are GLP-1molecules that are peptides containing one or more amino acidsubstitutions, additions or deletions, compared with GLP-1(7-36)amide.In one embodiment, the number of substitutions, deletions, or additionsis 30 amino acids or less, 25 amino acids or less, 20 amino acids orless, 15 amino acids or less, 10 amino acids or less, 5 amino acids orless or any integer in between these amounts. In one aspect of theinvention, the substitutions include one or more conservativesubstitutions. A “conservative” substitution denotes the replacement ofan amino acid residue by another, biologically active similar residue.Examples of conservative substitution include the substitution of onehydrophobic residue, such as isoleucine, valine, leucine, or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, and the like. The following table listsillustrative, but non-limiting, conservative amino acid substitutions.ORIGINAL RESIDUE EXEMPLARY SUBSTITUTIONS ALA SER, THR ARG LYS ASN HIS,SER ASP GLU, ASN CYS SER GLN ASN, HIS GLU ASP, GLU GLY ALA, SER HIS ASN,GLN ILE LEU, VAL, THR LEU ILE, VAL LYS ARG, GLN, GLU, THR MET LEU, ILE,VAL PHE LEU, TYR SER THR, ALA, ASN THR SER, ALA TRP ARG, SER TYR PHE VALILE, LEU, ALA PRO ALA

It is further understood that GLP-1 peptide variants include the abovedescribed peptides which have been chemically derivatized or altered,for example, peptides with non-natural amino acid residues (e.g.,taurine residue, β- and γ-amino acid residues and D-amino acidresidues), C-terminal functional group modifications such as amides,esters, and C-terminal ketone modifications and N-terminal functionalgroup modifications such as acylated amines, Schiff bases, orcyclization, such as found, for example, in the amino acid pyroglutamicacid.

Also included in the present invention are peptide sequences havinggreater than 50% sequence identity, and preferably greater than 90%sequence identity to (1) SEQ ID NOS: 1, 2, 3, 4; and (2) to truncatedsequences thereof. As used herein, sequence identity refers to acomparison made between two molecules using standard algorithms wellknown in the art. The preferred algorithm for calculating sequenceidentity for the present invention is the Smith-Waterman algorithm,where SEQ ID NO:1 is used as the reference sequence to define thepercentage identity of homologs over its length. The choice of parametervalues for matches, mismatches, and inserts or deletions is arbitrary,although some parameter values have been found to yield morebiologically realistic results than others. One preferred set ofparameter values for the Smith-Waterman algorithm is set forth in the“maximum similarity segments” approach, which uses values of 1 for amatched residue and −⅓ for a mismatched residue (a residue being eithera single nucleotide or single amino acid). Waterman, Bull. Math. Biol.46; 473 (1984). Insertions and deletions (indels), x, are weighted asX_(k)=1+k/3, where k is the number of residues in a given insert ordeletion. Id.

For instance, a sequence that is identical to the 42-amino acid residuesequence of SEQ ID NO: 1, except for 18 amino acid substitutions and aninsertion of 3 amino acids, would have a percent identity given by:[(1×42 matches)−(⅓×18 mismatches)−(1+ 3/3−indels)]/42=81% identity.

Also included in “GLP-1 molecules” of the present invention are sixpeptides in Gila monster venoms that are homologous to GLP-1. Theirsequences are compared to the sequence of GLP-1 in Table 1. TABLE 1Position        1 a. HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (NH₂) b.HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (NH₂) c.        DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (NH₂) d.HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (NH₂) e.HSDATFTAEYSKLLAKLALQKYLESILGSSTSPRPPSS (NH₂) f.HSDATFTAEYSKLLAKLALQKYLESILGSSTSPRPPS g.HSDAIFTEEYSKLLAKLALQKYLASILGSRTSPPP (NH₂) h.HSDAIFTQQYSKLLAKLALQKYLASILGSRTSPPP (NH₂)a = GLP-1(7-36)amide (SEQ. ID NO:4)b = exendin 3 (SEQ. ID NO:7).c = exendin 4 (9-39(NH₂) (SEQ. ID NO:8).d = exendin 4 (SEQ. ID NO:9).e = helospectin I (SEQ. ID NO:10).f = helospectin II (SEQ. ID NO:11).g = helodermin (SEQ. ID NO:12).h = Q⁸, Q⁹ helodermin (SEQ. ID NO:13).

Peptides (a, b, d, e, f, and g) are homologous at positions 1, 7, 11 and18. GLP-1 and exendins are further homologous at positions, 4, 5, 6, 8,9, 15, 22, 23, 25, 26 and 29. In position 2, A, S, and G arestructurally similar. In position 3, residues D and E (Asp and Glu) arestructurally similar. In positions 22 and 23, F (Phe) and I (Ile) arestructurally similar to Y (Tyr) and L (Leu), respectively. Likewise, inposition 26, L and I are structurally equivalent.

Thus, of the 30 residues of GLP-1, exendins 3 and 4 are identical in 15positions and equivalent in 5 additional positions. The only positionswhere major structural changes are evident are at residues 16, 17, 19,21, 24, 27, 28 and 30. Exendins also have 9 extra residues at theC-terminus.

Agonists of glucagon-like peptide that exhibit activity through theGLP-1(7-36)amide receptor have been described. See EP 0708179 A2; Hjorthet al., J. Biol. Chem. 269; 30121 (1994); Siegel et al., Amer. DiabetesAssoc. 57^(th) Scientific Session, Boston (1997); Hareter et al., Amer.Diabetes Assoc. 57^(th) Scientific Session, Boston (1997); Adelhorst etal., J. Biol. Chem. 269, 6275 (1994); Deacon et al., 16^(th)International Diabetes Federation Congress Abstracts, DiabetologiaSupplement (1997); Irwin et al., Proc. Natl. Acad. Sci. USA 94; 7915(1997); Mojsov, Int. J. Peptide Protein Res. 40; 333 (1992). Goke &Byrne, Diabetic Medicine 13; 854 (1996). Recent publications discloseBlack Widow GLP-1 and Ser.sup.2 GLP-1. See Holz & Hakner, Comp. Biochem.Physiol., Part B 121; 177 (1998) and Ritzel et al., J. Endocrinol. 159;93 (1998).

GLP-1 receptors are cell-surface proteins found, for example, oninsulin-producing pancreatic β-cells; the GLP-1(7-36) receptor has beencharacterised in the art. Methods of determining whether a chemical orpeptide binds to or activates a GLP-1 receptor are known to the skilledartisan and are preferably carried out with the aid of combinatorialchemical libraries and high throughput screening techniques.

GLP-1 molecule biological activity can be determined by in vitro and invivo animal models and human studies as is well known to the skilledartisan. GLP-1 biological activity can be determined by standardmethods, in general, by receptor-binding activity screening procedures,which involve providing appropriate cells that express the GLP-1receptor on their surface, for example, insulinoma cell lines such asRINmSF cells or INS-1 cells. See Mojsov, Int. J. Peptide Protein Res.40; 333 (1992) and EP 0708179 A2. Cells that are engineered to express aGLP-1 receptor also can be used. In addition to measuring specificbinding of tracer to membrane using radioimmunoassay methods, cAMPactivity or glucose dependent insulin production can also be measured.In one method, a polynucleotide encoding the GLP-1 receptor is employedto transfect cells so that they express the GLP-1 receptor protein.Thus, for example, these methods may be employed for screening for areceptor agonist by contacting such cells with compounds to be screenedand determining whether such compounds generate a signal (i.e., activatethe receptor). Other screening techniques include the use of cells thatexpress the GLP-1 receptor, for example, transfected CHO cells, in asystem to measure extracellular pH or ionic changes caused by receptoractivation. For example, potential agonists may be contacted with a cellthat expresses the GLP-1 protein receptor and a second messengerresponse (e.g., signal transduction or ionic or pH changes), may bemeasured to determine whether the potential agonist is effective.

Polyclonal and monoclonal antibodies can be utilized to detect purifyand identify GLP-like peptides for use in the methods described herein.Antibodies such as ABGA1178 detect intact GLP-1(1-37) orN-terminally-truncated GLP-1(7-37) or GLP-1(7-36)amide. Other antibodiesdetect the end of the C-terminus of the precursor molecule, a procedurethat allows one—by subtraction—to calculate the amount of biologicallyactive, truncated peptide (i.e., GLP-1(7-37)amide). Orskov et al.,Diabetes 42; 658 (1993); Orskov et al., J. Clin. Invest. 1991, 87; 415(1991).

The GLP-1 molecules of the invention that are peptides that can be madeby solid-state chemical peptide synthesis. Such peptides can also bemade by conventional recombinant techniques using standard proceduresdescribed in, for example, Sambrook & Maniaitis. “Recombinant,” as usedherein, means that a gene is derived from a recombinant (e.g., microbialor mammalian) expression system that has been genetically modified tocontain a polynucleotide encoding a GLP-1 molecule as described herein.

The GLP-1 molecule peptides of the present invention may be a naturallypurified product, or a product of synthetic chemical procedures, orproduced by recombinant techniques from prokaryotic or eukaryotic hosts(for example, by bacteria, yeast, higher plant, insect, or mammaliancells in culture or in vivo). Depending on the host employed in arecombinant production procedure, the polypeptides of the presentinvention are generally non-glycosylated, but may be glycosylated.

The GLP-1 like peptides can be recovered and purified from recombinantcell cultures by methods including, but not limited to, ammonium sulfateor ethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatography,and lectin chromatography. High performance liquid chromatography (HPLC)can be employed for final purification steps.

Particularly preferred GLP-1 molecules of the invention areGLP-1(7-36)amide, GLP-1(7-37), and exendin-4.

Formulation and Administration of GLP-1 for Therapeutic Treatment

Typically, a GLP-1 molecule of the invention will be administered in aparenteral formulation. In one preferred embodiment, the GLP-1 is in aliquid formulation. In a particularly preferred embodiment, the GLP-1molecule will be administered, at the first onset of symptoms, using asyringe comprising a liquid formulation of a pharmaceutically acceptableform of a GLP-1 molecule. Such a syringe, or “GLP-stick” can be used forself-administration of a GLP-1 molecule. Syringes forself-administration of drugs are well known in the art. See, e.g., U.S.Pat. Nos. 5,980,491 and 5,984,900. Also well known in the art aretuberculin-type syringes that are used for insulin injections.

Other well known methods for administration of a GLP-1 molecule to apatient suffering from UA or NQCN also can be employed in the methods ofthe invention. These administration methods include, but are not limitedto, subcutaneous or micropressure injection, external or implant pump,depot injection, and other types of prolonged application dispensingdevices. Other methods of administration, such as transdermal ortransmembrane administration, using patch or buccal means, also can beemployed. Oral administration also may be suitable. Pulmonaryadministration, such as inhalation, can also be employed.

Accordingly, embodiments of the invention include a GLP-1 moleculecontained in any type of syringe or device that is suitable forparenteral administration, also known as “kits.” These include, but arenot limited to, a pen-type syringe, an insulin-type syringe, a “pen”injector that delivers a metered dose, a needle-less injector, anexternal or implant pump, and a dry powder inhaler. Such kits compriseone or more doses of a GLP-1 molecule. Also included in the inventionare buccal or sublingual tablets comprising a GLP-1 molecule.

The amount of a GLP-1 molecule that should be administered will varyaccording to the severity of the conditions and the patient. Forself-administration using a GLP-stick, the total dose typically will be0.1-10.0 nmol/kg, preferably 1.5 nmol/kg. An advantage of usingGLP-1(7-36)amide is that high doses can be used without consequenthypoglycemia, because the action of GLP-1(7-36)amide is dependent onglucose levels. Therefore, doses of up to 10.0 nmol/kg can be usedwithout adverse effects. For continuous administration, levels of 0.1 to10.0 pmol/kg/min, preferably 1 to 4 pmol/kg/min, are used. Forcontinuous subcutaneous administration, levels of about 0.5 to 50pmol/kg/min or 0.5 to 50 pmol/kg/min, preferably about 1 to 10pmol/kg/min or 1 to 10 pmol/kg/min, are used.

The timing and dosage of a GLP-1 molecule, according to the methods ofthe invention, will depend on the nature of the condition being treated.As discussed here, a GLP-1 molecule may be administered as soon as thereis a symptom of cardiac distress, and the administration can becontinued, either continuously or an intermittent basis, for as long asnecessary. For example, the patient can self-administer a GLP-1 moleculeat the first symptom of cardiac distress, and a GLP-1 molecule canthereafter be administered during the time that the patient is intransit to the hospital, and continued during hospitalization, asnecessary. Thus, the GLP-1 molecule can be administered at the firstcardiac symptom, up until the time a Q-wave MI occurs. In the event thatsuch a QMI occurs following the administration of a GLP-1 molecule, thepretreatment of the patient with GLP-1 will ameliorate the tissue damagethat results from the MI. In alternative embodiments, the inventionincludes methods of administering GLP-1 at the first symptom of cardiacdistress, and continuing that administration during the time that theindividual suffers a QMI. In still further embodiments, the inventionincludes continuing administration of a GLP-1 molecule after theindividual has suffered a QMI. The administration of GLP-1 following aQMI will ameliorate the tissue damage that results from the QMI andsubsequent reperfusion-induced injury.

Pharmaceutically acceptable salts of a GLP-1 molecule also can be usedin the methods of the invention. Both organic and inorganic acidaddition salts can be employed, using acids that include, but are notlimited to, proprionic, succinic, lactic, malic, citric, acetic,benzoic, oxalic, carbonic, hydrochloric, hydrobromic, hydroiodic,sulfuric, and phosphoric.

A GLP-1 molecule, or a pharmaceutically acceptable salt thereof, can beformulated with a “pharmaceutically acceptable carrier or excipient,”which includes, for example, saline, buffered saline, dextrose, water,glycerol, ethanol, lactose, phosphate, mannitol, arginine, trehalose,and combinations thereof, and further includes agents that enhance thehalf-life in vivo of GLP-1, or a biologically active variant, analog, orderivative thereof, in order to enhance or prolong the biologicalactivity of the peptide or variant, analog, or derivative thereof.

Therapeutic Methods Using GLP-1

GLP-1 molecules, particularly GLP-1(7-36)amide, act to quickly suppressFFA levels and to optimize aberrant glucose metabolism in the heart, viaa variety of mechanisms. In particular, GLP-1(7-36)amide acts tosuppress glucagon secretion from pancreatic α-cells. GLP-1(7-36)amidehas no known serious adverse side effects and can be administered athigh doses without risking hypoglycemia or hyperglycemia. GLP-1molecules are ideal for optimizing glucose metabolism in a variety ofindividuals, including those with impaired glucose tolerance, and thosewith elevated or aberrant blood glucose levels that are induced bycertain conditions, such as stress-related cardiac conditions, andcardiac ischemia induced by UA or NQCN. The present inventioncontemplates treatment of individuals suffering from one or more of avariety of cardiac system disturbances or disorders, including but notlimited to UA and NQCN, which are described in this application. Inother embodiments, the inventive early stage treatment of UA canoptionally be continued during and after a QMI. In various embodimentsof the invention, these therapeutic methods include treatment ofindividuals with diabetes, including NIDDM, impaired glucose tolerance,and stress hyperglycemia.

In other preferred embodiments, the therapeutic methods of the inventiondo not include the treatment of an individual with type 2 diabetes (alsoknown as “Non-Insulin Dependent Diabetes Mellitus” or “NIDDM”). In stillother preferred embodiments, the therapeutic methods of the invention donot include treatment of an individual with any type of diabetes. Inadditional embodiments, the therapeutic methods of the invention do notinclude the treatment of an individual with impaired glucose tolerance.

In yet other embodiments of the present invention, the therapeuticmethods do not include the treatment of an individual suffering from aQ-wave MI. In additional embodiments, the therapeutic methods of thepresent invention do not include the treatment of an individualsuffering from a pathological Q-wave. The methods of the invention alsodo not include the treatment of an individual suffering from a STelevation, or ST elevation followed by T wave inversion. In otherembodiments, the present therapeutic methods do not include thetreatment of an individual having at least two values of serum CK-MB andCK that are at least two standard deviations above the normal range,10-16 hours after the onset of a symptom such a chest pain. In stillother embodiments, the present therapeutic methods do not includetreatment of an individual having at least two values of serum lactatedehydrogenase that are at least two standard deviations above the normalrange, and an isoenzyme pattern typical of QMI, within 48-72 hours afteronset of a symptom such as chest pain.

As discussed above, ischemic cardiac tissue switches to anaerobicglucose metabolism, from aerobic oxidation of FFA. Glucose oxidationconsumes less energy than FFA oxidation, and hence glucose oxidationpreserves borderline cardiac efficiency and increases cardiac efficiencyduring ischemia. Kantor et al., Am. J. Med. Sci. 318; 3 (1999). However,this switch to glucose metabolism during ischemia is usually incompletebecause glycolysis and glucose oxidation are inhibited in the presenceof insulin antagonism and high glucagon levels. In particular, excessblood FFA accumulate, and incomplete metabolism of FFA, or FFAoxidation, creates highly toxic free radicals that cause myocardialtissue damage.

Treatment with GLP-1 will ameliorate the adverse effects of cardiactissue ischemia. First, GLP-1 molecules promote glucose utilization bycardiac tissue, providing valuable energy. GLP-1 thus optimizes tissueutilization and metabolism of glucose, the major energy source duringcardiac ischemia. Second, GLP-1-molecule-mediated suppression ofglucagon will limit insulin antagonism and reduce circulating FFAs, thusfavoring glucose oxidation. These effects of GLP-1 are critical, becauseglucose oxidation consumes less oxygen than fatty acid oxidation.

Because of these beneficial effects of GLP-1 on glucose metabolism, thetherapeutic methods of the present invention will extend the time windowduring which thrombolytic therapy, such as TPA or angioplasty, will beeffective, following the first symptom of cardiac distress, such aschest pain lasting longer than 15 minutes, chest pain at rest, chestpain following minimal exertion, nausea, shortness of breath,palpitations, or dizziness. Thrombolytic therapy is known in the art fortreatment of coronary artery occlusion. See, e.g., Zaacks et al.,J.A.C.C. 33; 107 (1999). It also is known that thrombolytic therapy forQMI is only effective during a relatively brief time period followingQMI or strong clinical suspicion of QMI. Id.

TPA therapy generally is indicated when there has been (1) a diagnosedQ-wave MI, or (2) strong clinical suspicion of a Q-wave MI, accompaniedby ST segment elevation and an increase in troponins I and troponin Tassociated with QMI, and/or an increase in CK-MB that is associated witha QMI. Percutaneous transluminal coronary angioplasty (PTCA) istypically performed in a patient with a confirmed or suspected QMI,NQCN, or UA, when there is a strong suspicion of a developing adversecardiac event. PTCA is often used to treat any individual suffering fromACS. Accordingly, the therapeutic methods of the present invention willextend the time period, following cardiac distress, during whichangioplasty and/or TPA therapy will be effective.

It is known in the art that the PTCA procedure itself can result in therelease of small emboli, which can, in turn, cause cardiac ischemia whenthey become lodged in blood vessels. Accordingly, another embodiment ofthe invention is the treatment of a patient undergoing PTCA, with aGLP-1 molecule. The GLP-1 molecule will optimize metabolism, and henceameliorate or prevent the ischemic damage caused by the PTCA-inducedrelease of emboli. In one embodiment, the GLP-1 molecule is administeredcontinuously during the PTCA procedure. In other embodiments,administration of a GLP-1 molecule begins before the PTCA procedure, andcontinues during the procedure. In yet other embodiments, the GLP-1molecule administration is continued after the PTCA procedure iscompleted. In yet other embodiments, the invention includesadministering a GLP-1 molecule to a patient undergoing PTCA, wherein thepatient has not suffered a Q-wave MI. In other embodiments, the patienthas not exhibited a pathological Q-wave.

Acute Coronary Syndrome

It is an object of the present invention to provide a method of treatinga patient suffering from acute coronary syndrome, comprising treatmentof the patient with a therapeutically effective amount of a GLP-1molecule, such as GLP-1(7-36)amide. In particular, the present inventionprovides methods of treating patients suffering from unstable anginaand/or a non-Q wave cardiac necrosis.

In preferred embodiments, a patient suffering from unstable anginatreated using the inventive method has one or more of the followingconditions: (1) a blood troponin I level of no more than 0.4 ng/ml; (2)a blood troponin T level of no more than 0.1 ng/ml; (3) does not haveelevated CK-MB associated with QMI; (4) does not have elevated bloodlactate dehydrogenase associated with QMI; (5) does not have ST-segmentelevation; or (6) does not exhibit a pathological Q-wave.

In a preferred embodiment, the patient has the following symptoms: (1) ablood troponin I level of no more than 0.4 ng/ml; (2) a blood troponin Tlevel of no more than 0.1 ng/ml; (3) does not have elevated bloodcreatine kinase associated with QMI; (4) does not have elevated bloodlactate dehydrogenase associated with QMI; (5) does not have ST-segmentelevation; and (6) does not exhibit a pathological Q-wave.

It also is an object of the invention to provide a method for treating apatient suffering from non-Q-wave cardiac necrosis, wherein the patientdoes not have elevated blood CK-MB associated with QMI. In anotherembodiment, the patient has a blood troponin I level of no more than 0.4ng/ml. In another embodiment, the patient has a blood troponin T levelof no more than 0.1 ng/ml. In another embodiment, the patient does nothave ST-segment elevation. In another embodiment, the NQCN patient doesnot have elevated blood lactate dehydrogenase associated with QMI. Instill other embodiments, the patient does not have elevated bloodlactate dehydrogenase or elevated creatine kinase. In still anotherembodiment, the patient does not exhibit a pathological Q-wave.

The use of a GLP-1 molecule in the early stages of ACS, during UA and/orNQCN, will serve to optimize myocardial use of energy substrates, andwill limit ischemia-induced damage. Such use of GLP-1 will have theeffect of decreasing tissue damage, morbidity and mortality that isassociated with UA, NQCN, and Q-wave MI.

The invention also encompasses a method for treatment of an individualwith an established diagnosis of ACS (UA or NQCN) in whom there is—asye—-no evidence of an established Q-wave MI, in order to preserve andsalvage at-risk myocardial tissue in the ischemic and peri-ischemiczones. Such treatment will comprise the administration of intravenousGLP-1 by continuous infusion, in an appropriate liquid formulation, at adose of 0.1-10.0 pmol/kg/min, preferably 1.0-3.0 pmol/kg/min, forperiods of several hours and up to 10 days, preferably for one to threedays. Said continuous intravenous infusion of GLP-1 can be an isolatedtreatment, or in conjunction with the co-administration of intravenousglucose, as a continuous infusion of a 5-10% solution, and/or theco-administration of potassium, as a continuous infusion of a solutionof a suitable potassium salt (such as potassium chloride or potassiumacetate) that will supply sufficient potassium to maintain “normal”plasma potassium levels of about 4-5 mM. Typically the solution foradministration of potassium will be about 40 mM, but the skilled artisanwill recognize that any concentration of potassium can be used, so longas it supplies the desired dosage to the patient. Suitable rates foradministration of potassium are between about 40 and about 120μmol/kg/hr. Co-infusion with glucose is known to enhance and maintainthe insulinotropic drive of GLP-1; co-infusion of potassium is known tocorrect the hypokalemia that can potentially result from intracellularpotassium shifts that accompany insulin-mediated glucose uptake.

Patients Diagnosed With, or at Risk of Developing, IHD

It is known that individuals with diagnosed IHD, as evidenced inter aliaby previously documented ST segment changes or by clinical featuresdescribed elsewhere in this application, or those with a family historyof severe IHD, are at high risk for suffering from UA, NQCN, and Q-waveMI. Zaacks et al., J. Am. Col. Cardiol. 33; 107 (1999). According to oneaspect of the invention, a patient who has been previously diagnosedwith IHD, or is at strong risk for IHD, can then self administer GLP-1when he/she experiences a symptom that could be indicative of UA, NQCN,or future Q-wave MI. In other words, if such a patient experiences chestpain lasting longer than 15 minutes, chest pain at rest, or chest painfollowing minimal exertion, nausea, shortness of breath, palpitations,or dizziness, he/she can immediately administer a dose of GLP-1, using aGLP-stick. This may serve to prevent or ameliorate the damage that wouldbe caused by a future cardiac event. Thus, the invention includes amethod for treatment of a patient that has not suffered a Q-wave MI, buthas been diagnosed with IHD or is at strong risk for IHD, comprisingadministering to said individual GLP-1, after onset of one or more ofthe of the following symptoms: chest pain lasting longer than 15minutes, chest pain at rest, chest pain following minimal exertion,nausea, shortness of breath, palpitations, or dizziness.

The invention also encompasses a method for treatment of an individualwho previously has been diagnosed with likely IHD but with a normal ECG.Myerburg et al., “Electrocardiography”, In HARRISON'S PRINCIPALS OFINTERNAL MEDICINE (Isserbacher et al., eds.), 9th ed., pages 999-1011(McGraw Hill, Tokyo, 1980). The diagnosis of IHD is based on presence ofchronic, stable angina, elicited by exercise (also known as “exertionalangina”) and relieved by sublingual nitrates. Such an individual maypresent with symptoms consistent with a diagnosis of UA but have no ECGchanges. These symptoms include: chest pain (angina) of increasedfrequency, severity, or duration, provoked by mild exercise or at rest,where these symptoms are clearly different and more severe than thepreviously diagnosed chronic, stable angina, or where these symptomsappear in the context of recently diagnosed IHD. This typically occursabout 2 weeks to about 2 months before the onset of UA. When a symptomof cardiac distress appears, as described elsewhere in this application,GLP-1 should be self-administered at the earliest sign of such asymptom.

The invention additionally includes a method for treatment of a patientwith stable angina, comprising continuous administration of a GLP-1molecule. Such administration may be in the form of a patch suitable fortransdermal administration. Other suitable methods of administrationinclude continuous subcutaneous infusion, repeated subcutaneousinjection, and buccal, oral, or inhaled administration. Still furtherembodiments include administration using any other method ofadministration described in this application. The GLP-1 molecule willserve to benefit the coronary tissue through optimizing metabolism.

The invention also encompasses a method for treatment of an individualwho previously has been diagnosed as being at strong risk for IHD,because of a family history of severe IHD or a family history of IHD andassociated co-morbidities. “Family history of severe IHD” and “familyhistory of IHD and associated co-morbidities” are collectively referredto herein as “family history of IHD.” As used in this application, a“family history of severe IHD” denotes a history of a first-degreerelative who suffered one or more episodes of acute MI at age 50 oryounger, or died as a result of an acute MI or from post-MI heartfailure at age 60 or younger; “family history of IHD and associatedco-morbidities” denotes a history of angina or MI in a first-degreerelative plus the presence in the proband of one or more of thefollowing co-morbid conditions: diabetes, hypertension, hypercholeterolemia or hyperlipidemia, obesity, or history of smoking. When asymptom of cardiac distress appears, as defined above, GLP-1 should beself-administered at the earliest sign.

The invention also encompasses a method for treatment of an individualwho exhibits one or more symptoms (chest pain lasting longer than 15minutes, chest pain at rest, chest pain following minimal exertion,nausea, shortness of breath, palpitations, or dizziness), but whopreviously had no specific signs and symptoms of IHD—i.e., no previousepisodes of chest pain of cardiac origin (angina) and no previouslydocumented changes on ECG consistent with IHD (such as ST segmentdeviations and/or T wave inversions)—and who has no specific familyhistory of IHD, but who likely has underlying, undiagnosed IHD becauseof cardiac abnormalities not ascribed to other specific etiologies.These cardiac abnormalities include: (1) congestive heart failure, asevidenced, for example, by shortness of breath on mild exertion or atrest, exercise limitations, signs of pulmonary edema, and peripheraledema; (2) worsening heart murmur due to mitral regurgitation; or (3)evidence of cardiac conduction disturbances, such as left bundle branchblock on ECG, atrial or ventricular extrasystoles, atrial fibrillation,or other arrhythmias.

Therefore, the invention includes a method for the treatment of apatient, comprising administering to the patient a therapeuticallyeffective amount of a GLP-1 molecule, wherein said administration occursafter the onset of one or more of the following symptoms: (1) chest painlasting longer than 15 minutes, (2) chest pain at rest, (3) chest painfollowing minimal exertion, (4) nausea, (5) shortness of breath, (6)palpitations, or (7) dizziness, wherein said patient has one or more ofthe following cardiac abnormalities: (1) congestive heart failure, asevidenced, for example, by shortness of breath on mild exertion or atrest, exercise limitations, signs of pulmonary edema, and peripheraledema; (2) worsening heart murmur due to mitral regurgitation; or (3)evidence of cardiac conduction disturbances, such as left bundle branchblock on ECG, atrial or ventricular extrasystoles, atrial fibrillation,or other arrhythmias.

It is to be understood that the description, specific examples and data,while indicating exemplary embodiments, are given by way of illustrationand are not intended to limit the present invention. Various changes andmodifications within the present invention will become apparent to theskilled artisan from the discussion, disclosure and data containedherein, and thus are considered part of the invention.

EXAMPLES

Animal models may be used to test the efficacy of the administration ofGLP-1 to an individual with unstable angina, but without yet havingsuffered an actual infarction. Rat models and dog models have been foundto be particularly well suited for this purpose. In rats, GLP-1administered during the last 10 min. of a 25 min. ischemia period andthen throughout a 2-hour reperfusion period significantly reducedinfarct size (30%), and the rats also had significantly improvedhemodynamics. In dogs, administration of GLP-1 significantly reduced thestunning period, during reperfusion after a period of subcriticalischemia.

Example 1

Wistar rats were anesthetized with thiopentone sodium. The left anteriordescending (LAD) coronary artery was occluded. After 25 minutes ofocclusion, reperfusion was allowed for 2 hours. This animal model hasbeen described previously. Zacharowski, et al., Br. J. Pharmacol. 128;945-952 (1999).

GLP-1 (1.5 μg/kg/min) was infused into anesthetized rats (n=10),commencing 10 minutes prior to reperfusion and continuing throughout the2-hour reperfusion. Controls were sham operated with no occlusion (n=7),LAD occlusion+reperfusion+administration of saline (n=12), and LADocclusion and reperfusion with a buffer of 10 mM sodium acetate, 5.05%D-mannitol, pH 4.5, (“vehicle”) at 1.5 mL/kg/hour (n=10).

Following reperfusion, the coronary artery was reoccluded, and EvansBlue dye (4 ml, 2% w/v) was injected into the left ventricle of theheart via a right carotid artery cannula. Evans Blue stains perfusedmyocardium, while occluded vascular bed remains uncolored. Animals werethen killed by anesthetic overdose and the hearts were removed forexamination. Hearts were sectioned and the right ventricular wall wasremoved. The area at risk (pink) was separated from the non-ischemictissue (blue). The area at risk (pink) was then cut into smaller piecesand stained with p-nitroblue tetrazolium (NBT; 0.5 mg/ml) for 20 min. at37° C. In the presence of intact dehydrogenase enzyme systems (viablemyocardium), NBT forms a dark blue compound. Areas of necrosis lack theenzyme and remain unstained. Tissue was separated according to stainingand weighed to determine infarct size as a percentage of the area atrisk.

In rats receiving the saline infusion, the infarct size was 50±3% of thearea at risk. In rats receiving the vehicle infusion, the infarct sizewas 46±4% of the area at risk. In rats receiving the GLP-1 infusion, theinfarct size was 31±4% of the area at risk.

When compared with the vehicle group, infusion of GLP-1 caused astatistically significant (p<0.05) reduction in infarct size ofapproximately 33%. Thus, the systemic administration of GLP-1 can reducemyocardial infarct size even when administered after occlusion of acoronary artery and prior to onset of reperfusion.

Example 2

Two dogs were studied at baseline before, during, and for 6 hours aftera 10-minute complete left circumflex coronary (LCx) occlusion. Each dogunderwent occlusion/reperfusion in the presence and absence of GLP-1infusion for 24 hours, beginning 1 minute prior to reperfusion. GLP-1infusion enhanced the recovery of ventricular wall regional dysfunctionfollowing 10 minutes of coronary artery occlusion. The study shows thatthe recovery after ischemia and the reduced stunning in the presence ofGLP-1 are not due to increased coronary flow compared to controls, butpresumably reflect favorable changes in myocardial energetics.

1-47. (canceled)
 48. A method of treating a patient suffering from acutecoronary syndrome, comprising administering to the patient atherapeutically effective amount of an exendin.
 49. (canceled)
 50. Themethod of claim 48, wherein the exendin is exendin-3.
 51. The method ofclaim 48, wherein the exendin is administered in at least one dose. 52.The method of claim 48, wherein the exendin is administeredcontinuously.
 53. The method of claim 48, wherein glucose, a potassiumsalt, or a combination thereof, is co-administered with the exendin. 54.A method for treatment of a patient suffering from one or moreconditions selected from the group consisting of unstable angina,non-Q-wave cardiac necrosis, Q-wave myocardial infarction, acutemyocardial infarction, ischemic heart disease, and stable angina,comprising administering to said individual a therapeutically effectiveamount of an exendin, wherein said administration is after the onset ofone or more of the following symptoms: chest pain lasting longer than 15minutes, chest pain at rest, chest pain following minimal exertion,nausea, shortness of breath, palpitations, or dizziness
 55. The methodof claim 54, wherein the exendin is exendin-4.
 56. The method of claim54, wherein the exendin is exendin-3.
 57. The method of claim 54,wherein the exendin is administered in at least one dose.
 58. The methodof claim 54, wherein the exendin is administered continuously.
 59. Themethod of claim 54, wherein glucose, a potassium salt, or a combinationthereof, is co-administered with the exendin.
 60. A method forincreasing the time during which thrombolytic therapy will be effectivefollowing the first symptoms of cardiac distress, comprisingadministering a therapeutically effective amount of an exendin after theonset of one or more of the following symptoms: chest pain lastinglonger than 15 minutes, chest pain at rest, chest pain following minimalexertion, nausea, shortness of breath, palpitations, or dizziness. 61.The method of claim 60, wherein the exendin is exendin-4.
 62. The methodof claim 60, wherein the exendin is exendin-3.
 63. The method of claim60, wherein the exendin is administered in at least one dose.
 64. Themethod of claim 60, wherein the exendin is administered continuously.65. The method of claim 60, wherein glucose, a potassium salt, or acombination thereof, is co-administered with the exendin.